XXIV ICTAM, 21-26 August 2016, Montreal, Canada
MICROFLUIDIC GRANULAR AVALANCHES: A MODEL FOR GRAVITY DETECTION IN
PLANTS CELLS
Antoine Bérut∗1 , Hugo Chauvet1,2 , Bruno Moulia2 , Valérie Legué2 , Olivier Pouliquen1 , and Yoël Forterre1
1
Aix-Marseille Université, CNRS, IUSTI UMR 7343, 13453 Marseille cedex 13, France.
2
Clermont Université, Université Blaise Pascal, INRA, PIAF UMR 0547, F-63100 Clermont-Ferrand, France.
Summary We study the avalanche dynamics of a granular media composed of microparticles (melamine resine, 5.17 µm) in confined
microfluidics systems. This experimental set-up allows us to mimic the behaviour of statoliths in plants cells, which are thought to be
responsible for the plants’ response to gravity. Moreover, contrary to real plants (in-vivo) experiments, our avalanches are done in a fully
controlled environment, where the different roles of thermal fluctuations, non-Newtonian surrounding fluid, hydrodynamical and granular
effects can be investigated. Comparison of this biomimetic system with measurements in wheat shoots suggest that fluctuations induced by
cytoskeleton activity plays an important role in gravity detection by plants.
INTRODUCTION
Plants are able to adapt their growth to different directional environmental stimuli, such as light, gravity, moisture, etc.
In particular plants can bend in response to gravity, so that the roots grow downward and the shoots upward (figure 1a).
This gravitropism implies a way for plants to detect gravity, which has been widely studied by biologists [1]. The commonly accepted hypothesis states that the gravity detection is mediated by the movement of starch-accumulating amyloplasts
(statoliths), that sediment toward gravity in gravity sensing cells (statocytes), see figure 1b. However, the exact biological
mechanism for the intracellular detection and signalling is yet to be determined. Among the open questions, the role of biological activity in plants cells (e.g. actin cytoskeleton activity) remains unclear. Some studies states that it may enhance the
gravity detection by agitating the statoliths, which could avoid sensor saturation [2], some others states that, on the contrary,
plants with more cell activity are less sensitive to gravity [3], and finally a recent study considers the motion of statoliths as
pure Brownian motion without effect of cell activity [4].
Recently we have shown, with macroscopic experiments, that plant shoots are only sensible to the direction of gravity, and
not to its intensity [5]. This fact suggests that gravity sensing occurs mostly through detecting the position of a statoliths pile
inside the statocytes. In vivo microscopic observations of wheat cells have also shown that statoliths piles relax to a zero-angle
flat surface when the cells are subjected to a fast change of direction toward gravity (figure 1c). This behaviour is strikingly
different from what is expected for a classical granular media, where an angle of repose exists. A key question is to know if
the Brownian thermal agitation can explain this behaviour, or if the biological activity is needed to observe such a relaxation.
(a)
(b)
(c)
Figure 1: a) Images of a wheat coleoptile bending during 2 h of growth in response to gravity. b) A wheat coleoptile statocyte
containing statoliths, observed with a ×40 microscope objective. c) Relaxation of the angle of the statoliths pile’s free surface
after a rotation of 70◦ , averaged over several statocytes.
RESULTS
In first approximation, statoliths can be considered as a passive Brownian granular medium. However, the dynamics inside
the statocytes is very complex: the statoliths are very confined, the surrounding fluid is non-Newtonian, and the plants cells
show biological non-thermal activity. Moreover, in vivo experiments are difficult, due to the relatively short life-time of plants
∗ Corresponding
author. Email: [email protected]
cells when placed between glass slide and cover slip for microscopic observations. To avoid these problems, we designed a
biomimetic artificial system, where statoliths-like particles are embedded in plant cells-like microfluidic devices.
Our biomimetic devices are made of a matrix of small containers with dimensions close to those of wheat statocytes
(approximatively 100 µm × 30 µm, and 50 µm depth). They are fabricated using classical microfluidic techniques: a negative
master mould is created with SU-8 photoresist and a photolithography mask, then negative replicas are made using PDMS
or agar-agar. The artificial statoliths are melamine resine microparticles (5.17 µm in diameter), with a density of 1.51 g/cm3 ,
which is close to starch density. The particles can be dispersed in bidistilled water (for simplicity) or other fluids with close
density (to change the rheological properties). Hence, the particles show the same Brownian behaviour as real statoliths,
because they have the same dimensionless number ζ ∝ kB T /∆ρR4 g (where kB is the Boltzmann factor, T the temperature,
∆ρ the difference of the particle’s and fluid’s densities, g the gravity on Earth, and R the particle’s radius). ζ compares the
strength of the thermal activity acting on the particle to it’s own weight.
The system is observed with a customised microscope, placed horizontally, so that the gravity vector is contained in the
observation plane (whereas gravity is perpendicular to the observation plane in an usual microscope). A picture of an in vivo
sample is shown figure 2a, in comparison with one of our micro-fluidic devices in figure 2b. The sample is held on a rotation
stage, which allows us to change rapidly its direction with respect to gravity. We observe the avalanches and subsequent
relaxations of the microparticles piles in the confined geometry of the cells. The images recorded are then analysed to extract
the fluctuations of the free surface, and in particular the mean angle between this surface and the gravity direction. The fact
that we use a matrix of separate cells allows us to increase the statistics in a single experimental run.
(a)
(b)
Figure 2: Comparison between in vivo observation and artificial biomimetic device. a) A wheat coleoptile tissue with statocyte
containing statoliths, observed with a ×10 microscope objective. b) Agar-agar matrix of cells containing melamine resin
microparticles, observed with a ×10 microscope objective.
PERSPECTIVES
The use of artificial microfluidic devices with geometrical properties close to those of real plants cells allows us to study
the motion of statolith-like particles in a fully controlled system, where rheological properties and thermal agitation are known.
Preliminary results suggests that Brownian motion without cell activity is not enough to retrieve the relaxation observed with
statoliths. By changing our particles’ radius, we will be able to increase the influence of the thermal fluctuations to check for
the equivalent agitation needed for gravity detection in plants cells.
This work was supported by the European Research Concil (ERC) under the European Unions Horizon 2020 research and innovation
programme (grant agreement No. 647384) and by the French National Research Agency (ANR-13-BSV5-0005-01).
References
[1] M. Morita, Directional Gravity Sensing in Gravitropism, Annual Review of Plant Biology, vol. 61: 705-720 (2010).
[2] G. Leitz, B.-H. Kang, M. E.A. Schoenwaelder, and L. A. Staehelin, Statolith Sedimentation Kinetics and Force Transduction to the Cortical Endoplasmic Reticulum in Gravity-Sensing Arabidopsis Columella Cells, The Plant Cell, vol. 21, no. 3, 843-860 (2009).
[3] M. Nakamura, M.Toyota, M. Tasaka, and M. Morita, An Arabidopsis E3 Ligase, SHOOT GRAVITROPISM9, Modulates the Interaction between
Statoliths and F-Actin in Gravity Sensing,The Plant Cell, vol. 23, no. 5, 1830-1848 (2011).
[4] Z. Zheng J. Zou, H. Li, S. Xue, Y. Wang, and J. Le, Microrheological Insights into the Dynamics of Amyloplasts in Root Gravity-Sensing Cells,
Molecular Plant, 8 , 660-663 (2015).
[5] Article in preparation.